Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools, but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious, and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique

A non-surgical approach for male germ cell mediated gene transmission through transgenesis

Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers

A Study of Differential Expression of Testicular Genes in Various Reproductive Phases of Hemidactylus flaviviridis (Wall Lizard) to Derive Their Association with Onset of Spermatogenesis and Its Relevance to Mammals.

Testis of Hemidactylus flaviviridis, commonly known as Indian wall lizard, displays a lack of cellular and metabolic activity in regressed phase of testis during non-breeding season of the year. Retracted Sertoli cells (Sc), fibroid myoid cells and pre-meiotic resting spermatogonia are observed in such testis. This situation is akin to certain forms of infertility in men where hormone supplementation fails to generate sperm despite the presence of Sc and germ cells (Gc) in testis. In testis of lizard, spermatogenesis is reinitiated upon increased level of hormones during appropriate season (phase of recrudescence). Study of genes associated with generation of sperm, from regressed adult testis in lizard, may provide valuable information for understanding certain forms of male idiopathic infertility. Subtractive hybridization using testicular RNA obtained from the regressed and active phases of lizard reproductive cycle led to identify eight partial mRNA sequences that showed sequence homology with mice genes. We further evaluated the gene expression prolife by real-time PCR in three different reproductive phases of H. flaviviridis: regressed (pre-meiotic), recrudescent (meiotic) and active (post meiotic), for comparison with the corresponding testicular phases found in testis of 5 days (pre-meiotic), 20 days (meiotic) and 60 days (post-meiotic) old mouse. This is the first report where genes associated with progression of spermatogenesis during active phase, which follows a regressed state of adult testis, were identified in lizard and found to be conserved in mouse. Six important genes, Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b were found to be strictly associated with active spermatogenesis in both mouse and lizard. Factors interfering with the expression of any of these genes may potentially abrogate the process of spermatogenesis leading to infertility. Such information may shed light on unknown causes of idiopathic male infertility

Advantages of pulsatile hormone treatment for assessing hormone-induced gene expression by cultured rat Sertoli cells

In response to various hormonal (follicle-stimulating hormone [FSH] and testosterone [T]) and biochemical inputs, testicular Sertoli cells (Sc) produce factors that regulate spermatogenesis. A number of FSH- and T-responsive Sc-specific genes, necessary for spermatogenesis, have been identified to date. However, the hormone-induced in vitro expression pattern of most of these genes is reported to be inconsistent at various time points in primary rat Sc cultures. As a matter of convenience, cultured Sc are constantly exposed to hormones for a few hours to days in the reported literature, although Sc are exposed to pulsatile FSH and T in vivo. The major aim of the present study is to evaluate the advantage, if any, of the in vitro administration of pulsatile hormone (FSH and T in combination) treatment on gene expression of cultured Sc as compared with that of constant hormone treatment. Pulsatile treatment (a 30-min hormonal exposure every 3 h) mimicking the in vivo condition reveals a more prominent effect of hormones in augmenting gene expression as compared with constant treatment. Our results indicate that the expressions of Stem cell factor (Scf, only responsive to FSH), Claudin11 (only responsive to T) and Transferrin (both FSH- and T-responsive) mRNAs are significantly higher at 12 h upon pulsatile treatment than upon constant hormonal treatment. Maximal expression of relevant genes because of pulsatile treatment with hormones suggests that this protocol provides a more suitable premise for assessing hormone-induced gene expression in isolated Sc than one involving constant exposure to hormones

Flowchart of subtractive hybridization protocol for lizard testicular samples.

<p>Schematic representation depicting method of subtractive hybridization between RNA isolated from testes of two different reproductive phases (active and regressed phase) of wall lizard.</p

List of partial transcript sequences of lizard and their sequence similarity with homolog-genes of mice.

<p>List of partial transcript sequences of lizard and their sequence similarity with homolog-genes of mice.</p

Comparative analysis of mRNA expression of selected genes in testis of wall lizards and mice.

<p>Shows level of mRNA expression of <i>Hk1</i> (A& I), <i>Nme5</i> (B& J), <i>Akap4</i> (C& K), <i>Arih1</i> (D &L), <i>Rassf7</i> (E & M), <i>Tubb4b</i> (F & N), <i>Bco2</i> (G & O) and <i>Hmgb1</i> (H & P) in three reproductive phases of wall lizard and three developmental stages of mice testis respectively. Normalization of gene expression was done with reference gene <i>Gapdh</i> for wall lizard and <i>Ppia</i> for mouse. Correlation between relative fold change in genes expression and testis weight in wall lizard and mice for genes<i>Hk1</i> (Q), <i>Nme5</i> (R), <i>akap4</i> (S), <i>Arih1</i> (T), <i>Rassf7</i> (U) and <i>Tubb4b</i> (V). <i>Bco2</i> (W), and <i>Hmgb1</i> (X) were analyzed. A solid or broken line in scatter plots denotes linear regression. Open circle with dotted line represents mice and closed circle with solid line represents wall lizard relative gene expression with respect to testicular wt. in percentage. Rg-T, Rc-T, Ac-T are testes from regressed, recrudescent, and active phase of wall lizard. 5d-T, 20d-T, and 60d-T are testes from 5 days, 20 days and 60 days old mice. *p<0.05, **p<0.01 and ***p<0.001 represents the comparison of Rg-T with Rc-T and Ac-T in wall lizards and 5d-T with 20d-T and 60d-T in mice. ^#p<0.05, ^##p<0.01 and ^###p<0.001 represents the comparison of Rc-T with Ac-T in wall lizards and 20d-T with 60d-T in mice.</p

Predicted interaction among proteins.

<p>Probable interactome of different proteins, of which mRNA were differentially expressed in different testicular phase of wall lizard mice. Probable interacting partner of gene products, <i>Hk1</i> (A), <i>Nme5</i> (B), <i>Akap4</i>(C), <i>Arih1</i> (D), <i>Rassf7</i> (E), and <i>Hmgb1</i> (F) with other proteins as per STRING database.</p

Summarized result of subtractive hybridization analysis.

<p>Distribution of total partial sequences, obtained from subtractive hybridizations analysis between testicular RNA isolated from regressed and active phase of wall lizard.</p

Heat map analysis of gene expression between lizards and mice.

<p>Heat map analysis showing differential gene expression pattern in pre-meiotic, meiotic and post-meiotic testes of wall lizard and mice. The relative fold change in gene expression for genes <i>Hk1</i>, <i>Nme5</i>, <i>Akap4</i>, <i>Arih1</i>, <i>Tubb4b</i>, <i>Rassf7</i>, <i>Bco2</i>, and <i>Hmgb1</i> were compared in corresponding testes of wall lizards and mice. Color from red to green indicates high to low expression.</p